The differentiation of arterial vs. venous endothelial cells (EC) occurs early during vertebrate cardiovascular development, and appears to involve interplay of both intrinsic genetic factors and environmental stimuli. Biomechanical forces generated by blood flow are strong modulators of the endothelial phenotype in health and disease. Our preliminary studies in vitro have revealed that venous EC can be modulated towards an arterial identity when exposed to a biomechanical stimulus that simulates components of the arterial circulation. In addition, a mouse model of venous arterialization has demonstrated the expression of arterial identity markers in EC of veins exposed to an arterial circulation. Thus, we hypothesize that hemodynamic forces can act as cell differentiation stimuli controlling the establishment and maintenance of endothelial arterial and venous identities. To test this hypothesis we propose three Specific Aims. In Aim 1, the transcriptional programs evoked when cultured human venous EC are exposed to arterial shear stress waveforms will be characterized and compared with those observed in EC freshly isolated from human arteries and veins. In Aim 2, the role of Notch signaling in flow-mediated venous-to-arterial endothelial differentiation will be investigated utilizing gain-of-function and loss-of-function mutants of Notch and its downstream effectors. In Aim 3, a novel mouse model will be used to test the in vivo relevance of Notch and two of its transcriptional targets, the bHLH transcription factors Hey-1 and Hey-2, in venous arterialization. Our long-term goal is to understand the flow-mediated molecular and cellular mechanisms that control arterial vs. venous specification in the vascular system. These studies should provide important mechanistic insights into the control of endothelial identity by hemodynamic forces, and may suggest new therapeutic strategies for diseases in which dysregulation of this process leads to serious pathologies, including saphenous vein bypass graft failure and arterio-venous malformations.